In both research applications and clinical diagnosis, a known technique for determining the presence of a particular nucleotide sequence (the "target nucleotide sequence") in either RNA or DNA is to perform a nucleic acid hybridization assay. In such an assay, a nucleotide probe, typically an oligonucleotide, is selected having a nucleotide sequence complementary to at least a portion of the target nucleotide sequence. Typically, the probe is labeled to provide a means whereby the presence of the probe can be readily detected.
When the labeled probe is exposed to a sample suspected of containing the target nucleotide sequence, under hybridizing conditions, the target sequence will hybridize with such a labeled probe. The presence of the target sequence in the sample can then be determined qualitatively or quantitatively, usually after separating hybridized and non-hybridized probes and determining the presence or amount of the labeled probe which hybridized to the test sample.
Prior methods for linking a label to a nucleotide probe have generally utilized a single label attached to a nucleotide monomeric unit, and then incorporating one or more of the nucleotide monomeric units into the probe. For example, analogs of dUTP and UTP containing a biotin moiety have been chemically synthesized and incorporated into polynucleotides (P. R. Langer et al., Proc. Nat. Acad. Sci. USA 78:6633 (1981)). Such biotin-labeled nucleotides may then be incorporated into nucleic acid probes of biological or synthetic origin.
Other methods for labeling nucleotide probes have been proposed which allow labels to be randomly linked to nucleotides in a nucleotide multimer. Numerous proposals have been made for incorporating multiple modified nucleotides or non-nucleotide monomeric units into oligonucleotides with a view towards enhancing the detectability of the labeled probe and the target nucleotide sequence.
However, it has been demonstrated that use of such labeled nucleotides in a probe can reduce the stability of the hybrid formed with a target nucleotide sequence, particularly when multiple labels are present. Such reduced hybrid stability has been demonstrated for nucleic acid probes of biological origin possessing multiple biotin moieties, for synthetic oligonucleotides possessing multiple fluorescein labels, as well as for synthetic oligonucleotides possessing biotin and fluorescein labels.
In addition, derivatives of nucleotide linking phosphate groups have been disclosed, the nucleophilic moiety of which can be labeled following their incorporation into an oligonucleotide. However, such compounds, being based on nucleotide derivatives, would be expected to exhibit some of the disadvantages discussed above for nucleotide based derivatives.
More recently, 2-amino-1,3-propanediol structures have been used to label oligonucleotides with reporter groups (Nelsen, P. S. et al., Nuc. Acids Res. 20:6253 (1992)). However, these structures appear to demonstrate low coupling efficiency, and thus low yield of labeled oligonucleotides which furthermore must be carefully purified before they can find use as probes for target sequences.
Thus it is considered desirable to provide a non-nucleotide reagent which demonstrates high coupling efficiency and thus provides higher yield of labeled oligomer.
Furthermore, it is also considered desirable to provide such a reagent which will allow the resultant oligomers to anneal and hybridize with efficiencies approaching those of oligomers which contain only native nucleotide monomeric units.